This project involves a comprehensive approach toward the design and implementation of a therapy that will allow for solid organ allotransplants to be performed with a reduced requirement for maintenance immunosuppression. Therapies are evaluated in non-human primates (NHPs) for translation into clinical trials. The primary pre-clinical model is the rhesus monkey kidney (or in some cases skin) transplant model. Hypothesis tested. Treatment with biologicals designed to interrupt costimulatory pathways at the time of primary alloantigen exposure will prevent acute allograft rejection and induce durable allospecific tolerance. Problem and Significance. Several rodent studies have suggested that interruption of many T-cell costimulatory pathways induces a sustained state of allograft acceptance. Lasting, donor-specific allograft acceptance has been achieved without recipient morbidity using brief perioperative treatments with antibodies specific for molecules in the CD28-B7 and CD40-CD154 pathways. If this were true in humans, costimulation-based treatments would greatly reduce the expense and morbidity of transplantation. However, rodent models are overly permissive and infrequently reflect the clinical applicability of novel therapies. Furthermore, human-specific biologics must typically be evaluated in cross-reactive primate models prior to use in humans. These studies thus test the efficacy of costimulation-specific therapies in models that determine the generalizability of rodent work, and thus support translation to humans when appropriate. We have built upon previous work in this model with costimulation-blockade to demonstrate that treatment with the humanized CD154 specific monoclonal antibody (Mab) hu5c8 prevents renal and skin allograft rejection and has limited utility in reversing established rejection in NHPs. We have also demonstrated that donor specific transfusion (DST) synergizes with anti-CD154 in primates as does the mTOR inhibitor sirolimus. We have therefore initiated trials in NHPs designed to optimize the timing, dose, route and source of DST. In primates, we now have evidence that portal venous infusion of unfractionated bone marrow given preoperatively in combination with sirolimus, but not tacrolimus, greatly delays rejection with no other immunosuppressive therapy. We are now beginning trials combining the optimized DST regimen with the anti-CD154 Mab IDEC-131 to see if tolerance can be reproducibly achieved in this rigorous preclinical model. One realization coming from the CD154 therapy literature is that CD154 is expressed not only on T cells, but also on activated platelets. Indeed, one drawback of anti-CD154 has been its association with thromboembolic events, perhaps related to the interaction with platelets. We have therefor initiated studies in rodents to determine whether platelet derived CD154 is relevant to allograft rejection. Using combinations of CD154 knock out mice, adoptive platelet transfusions, recombinant CD154 and cardiac transplants, we have shown that platelet derived CD154 is indeed sufficient to induce rejection without any not platelet source of this molecule. This is the first demonstration of a non-lymphocyte derived axis that mediates alloimmunity. The results of these fundamental findings are being prepared for submission. To complement clinical studies in depletional induction we have also developed a novel polyclonal depleting antibody that has better T cell depleting ability and contains enhanced blockade of costimulation molecules while leaving molecules vital for regulatory function relatively spared. This agent is now being tested in vitro and prepared for initial in vivo studies in primates. Ischemia reperfusion injury is a know stimulator of alloimmunity. Through a collaboration with Mark Gladwin, we are providing NHP model support for the evaluation of Nitrite as a method for limiting vasospastic renal injurry and acute tubular necrosis following renal transplantation.